The present invention relates to a method for diagnosing Alzheimer""s disease.
Alzheimer""s disease is a neurodegenerative dementia characterized by a loss of cortical neurons associated with xcex2-amyloid plaques, of neurofibrillary tangles and, in most cases, an amyloid angiopathy. It is strongly suspected that there is a genetic influence in the aetiology of Alzheimer""s disease (WO 94/01772).
This genetic component has been brought to the fore over many years by indirect observations which suggest that the disease is inherited in an autosomal dominant fashion with an age-dependent penetrance in order to explain certain familial forms of Alzheimer""s disease. Recent molecular genetic studies have enabled putative genes for Alzheimer""s disease to be isolated by looking for chromosome-specific polymorphic genetic markers (Bird et al., 1989, Neurobiology of Aging 10, 432-434).
Four chromosomal localizations have been described as being involved: three on chromosomes 1, 14 and 21 in the early onset familial forms (age at onset under 60 years), and one on chromosome 19 in the late onset familial and sporadic forms. Two linkage studies have suggested that the chromosomal region 19q13.2 was associated with late onset familial forms of Alzheimer""s disease (Pericak-Vance et al, Am. J. Hum. Genet. (1991), 48, 1034-1050). Within this chromosomal region, the group of genes for apolipoproteins (APO) E-CI-CIxe2x80x2-CII is a candidate zone. Among the products of these genes, apolipoprotein E (APOE) is involved especially in the nervous system APOE is present in the senile plaques and possesses a binding affinity for the peptide Axcex2. APOE is characterized by three major alleles xcex52, xcex53, xcex54. Strittmatter et al. (Proc. Natl. Acad. Sci. (1993) 90, 177-181) have described an increased frequency of the xcex54 allele of the APOE gene in the late onset familial forms of Alzheimer""s disease. This observation has been confirmed for the familial forms (Corder et al., Science (1993), 261, 921-923) and the sporadic forms of Alzheimer""s disease (Corder et al., Science (1993), 261, 921-923; Saunders et al., Neurology (1993), 13, 1467-1472).
FR-2 716 894 describes a method which makes it possible to prognosticate, for a given disease, the risks of developing Alzheimer""s disease with respect to the general population. This method is based on the detection of the xcex54 alleles of the APOE gene, of the short alleles of the marker D19S178 and of the long alleles of the APO CII gene, all localized on chromosome 19.
Several hypotheses make it possible to explain this phenomenon.
Recent studies by the inventors, who are the authors of the present invention, now confirm the hypothesis of the existence of at least one other functional mutation in the 19q13.2 region.
Indeed, in addition to a functional effect specific to the polymorphism of apolipoprotein E, the studies by the inventors show differences in levels of expression which are significant in sick subjects compared with healthy controls, which indicates that one or more mutations in the regulatory regions of the APOE gene are involved in the onset of Alzheimer""s disease. Furthermore, relative differences in expression are found in the controls.
The inventors have more particularly identified a new polymorphism in the region of the promoter of the gene encoding the apolipoprotein E protein found in a potential binding site for the Th1/E47cs transcription factors.
For the determination of this polymorphism, the sequence described by Paik et al. (1985, Proc. Natl. Aca. Sci., vol. 82, p. 3447) will be taken as reference.
This polymorphism has been called by the inventors Th1/E47cs for Th1/E47cs consensus, since it is situated in a consensus sequence for binding of the Th1/E47cs transcription factor.
The mutation identified is characterized by a Thymine to Guanine substitution (Gxe2x86x92T) in the sequence:
GGGTGTCTGT(or G)ATTACTGGG,
G being the most frequent allele in the normal population.
The alleles corresponding to this polymorphism are called hereinafter T (when the base is Thymine) or G (when the base is Guanine).
The determination of the allele can be carried out after PCR amplification of the DNA region comprising this polymorphism:
either by creating in one of the PCR primers a cleavage site for a restriction enzyme not existing in individuals carrying one of the alleles,
or by a hybridization technique using oligonucleotide probes specific for the alleles.
The inventors have studied the influence of the Th1/E47cs polymorphism on the expression of the alleles of the APOE gene and demonstrated an increase in the risk of developing Alzheimer""s disease associated with the T allele of Th1/E47cs, specific to this allele and not due to the xcex54 allele.
Furthermore, the Th1/E47cs polymorphism modulates the risk associated with the xcex54 allele in individuals with the xcex53/xcex54 genotype, the individuals with the GT genotype having an increased risk of developing Alzheimer""s disease compared with the individuals homozygous for the Th1/E47cs polymorphism. Reinforcing this observation, the authors have considered that the combination of the T allele of Th1/E47cs with the xcex54 allele on the same chromosome corresponds to the most unfavourable combination.
The subject of the invention is thus a method for diagnosing Alzheimer""s disease, comprising the identification of one or more mutations in the genomic DNA region for regulating the expression of the apolipoprotein E gene, inducing a modification of the expression of the apolipoprotein E gene relative to a control population or a modification of the relative expression of the alleles of the apolipoprotein E gene.
For the purposes of the present invention, diagnosis is understood to mean the confirmation of a mutation in the regulatory region of the APOE gene in a patient whose clinical picture signals a symptomatology which may be attributed to Alzheimer""s disease, or alternatively an increased probability in subjects of developing Alzheimer""s disease relative to the population as a whole, the increase in probability being statistically significant.
The chromosomal DNA region for regulating the APOE gene is broadly defined as being the chromosomal region 19q13.2 other than the region encoding apolipoprotein E (Human Molecular Genetics, 1994, vol. 3, No. 4, 569-574).
Advantageously, the chromosomal DNA region in which one or more mutations are identified is situated between the marker D19S178 and the APOCII gene, and comprises more particularly the introns and the flanking regions of the APOE gene, extending over a distance of 5 kb upstream and downstream of the APOE gene.
The subject of the invention is more particularly a method for diagnosing Alzheimer""s disease comprising the identification of at least one mutation in the promoter of the APOE gene, situated at 186 bases from the TATA box of this gene, the mutation consisting more particularly of the replacement Txe2x86x92G in the sequence defined above.
More particularly, the method consists in testing for one or more mutations in the region of the promoter of the gene encoding apolipoprotein E, existing in particular in a potential binding site for the Th1/E47 transcription factors.
The subject of the invention is also a method for diagnosing Alzheimer""s disease comprising a determination of the genotype of apolipoprotein E and the test for a mutation of the type described above in the regulatory region of the APOE gene.
In accordance with this diagnostic method, the presence of at least one xcex54 allele of apolipoprotein E conjointly with the existence of a mutation in the regulatory region of the APOE gene, in particular the mutation defined above inducing a modification of the expression of the APOE gene or a relative difference in expression of the alleles of apolipoprotein E where appropriate, will range towards the diagnosis of Alzheimer""s disease in patients whose clinical picture presents a symptomatology which can be attributed to Alzheimer""s disease or will make it possible to classify subjects in good health in a category with an increased risk of developing Alzheimer""s disease.
Relative difference in expression of the alleles is understood to mean a difference in expression of one allele relative to another, independently of the absolute level of expression of the gene.
The test for the xcex54 allele is done by any appropriate method based on the presence of an ARG residue at position 112 of apolipoprotein E for the xcex54 allele, of a CYS residue at position 158 for the xcex52 allele, relative to the residues CYS and ARG in these positions for the xcex53 isoform, which is the most widespread.
The identification of an additional mutation in the regulatory regions of the APOE gene as defined above is carried out by any appropriate method, in particular a method for diagnosing Alzheimer""s disease comprising the identification of at least one mutation in the promoter of the gene encoding APOE, situated at 186 bases from the TATA box of this gene.
The presence of at least one xcex54 allele of the APOE gene, of at least one short allele of the marker D19S178 and of at least one long allele of the APO CII gene as described in FR-2,716,894 and the existence of at least one mutation in the regulatory region of the APOE gene will strongly contribute towards orienting the diagnosis of Alzheimer""s disease in symptomatic subjects or will constitute a substantial risk factor in asymptomatic subjects.
Since the mutation(s) involved in Alzheimer""s disease are responsible for a variation in the relative expression of the alleles of the APOE gene in the brain, it is also possible to determine, instead of or in addition to a mutation in the regulatory regions of the APOE gene which are defined above, the level of expression of the APOE gene and to compare this level of expression to that of the general population.
The diagnostic method based on the determination of the level of expression of apolipoprotein E is particularly advantageous in heterozygous subjects, in particular carrying the xcex54 allele. In these subjects, the diagnostic method for the purposes of the invention advantageously comprises the determination of the level of expression of the xcex54 allele relative to the xcex52 or xcex53 allele.
In the same manner, the diagnostic method in the individuals with the xcex52/xcex53 genotype advantageously comprises the determination of the level of expression of the xcex52 allele relative to the xcex53 allele.
An increase or a significant decrease, respectively, in the level of expression/transcription of the xcex54 allele in a heterozygous subject xcex54xcex52 or xcex54xcex53 and of the xcex52 allele in a heterozygous subject xcex52xcex53, will orient the diagnosis towards a dementia of the Alzheimer type, if moreover the subject presents a clinical picture evoking the symptomatology of Alzheimer""s disease. In a subject not presenting apparent clinical signs, the increase or the decrease in expression of the xcex54 and xcex52 alleles, respectively, will be an indication of an increased probability in the subject of subsequently developing Alzheimer""s disease.
The determination of the level of expression of the APOE gene and more particularly of the xcex54 and xcex52 alleles of the APOE gene is advantageously carried out by measuring the relative level of the mRNA for the APOE gene either by establishing the ratio of transcription of the xcex54 allele relative to the xcex52 or xcex53 allele in the case of individuals with the xcex54xcex52 and xcex54xcex53 genotypes, or by establishing the ratio of transcription of the xcex52 allele relative to the xcex53 allele in the case of individuals with the xcex52xcex53 genotype.
The measurement of the level of transcription is carried out following the extraction of mRNA from biopsy tissues or from cells in cultures and amplification by RT-PCR (reverse transcription polymeric chain reaction), with the aid of appropriate primers specific for the allele for which it is desired to measure the level of expression.
The tissue used is for example derived from a biopsy of cerebral tissue, in particular of frontal lobes, thus making it possible to measure the level of expression of the alleles of APOE in the brain. It is also possible to determine the level of transcription of the mRNAs for APOE in lymphocytes or fibroblasts in cell culture. In general, it will be possible to determine the level of transcription of the alleles of APOE in any tissue capable of exhibiting a variation in the percentage of expression of an allele relative to another between the patients and those who are sick. Likewise, this method can also be applied for the development and the use of cellular or animal models using the alleles of APOE.
The ratio of the expression of the alleles of the APOE gene is determined in the following manner:
a) the cDNA is subjected to a PCR amplification in the presence of primers permitting the specific amplification of at least one polymorphic sequence of the alleles;
b) the amplified DNA is subjected to the action of at least one restriction enzyme, permitting the differentiation of the alleles;
c) the DNA fragments are separated;
d) the quantity of fragments obtained is evaluated by means of a marker emitting a detectable signal;
e) the initial ratio in the different alleles is determined by the following formula:             N              o        ⁢                  xe2x80x83                ⁢        allele1                            N                  o          ⁢                      xe2x80x83                    ⁢          allele1                    +              N                  o          ⁢                      xe2x80x83                    ⁢          allele2                      =            A      ⁢              xe2x80x83            ⁢              α        allele1        xe2x80x2                            A        ⁢                  xe2x80x83                ⁢                  α          allele1          xe2x80x2                    +              α        allele2        xe2x80x2            
in which No is the initial number of DNA molecules,
A is the coefficient of proportionality which makes it possible to correct the size difference between the different restriction fragments and is equal to the ratio of the lengths of restriction fragments characteristic of each allele,
and xcex1xe2x80x2 is determined
either by the following formula:       α    xe2x80x2    =            O      ⁢              xe2x80x83            ⁢              D        max                    K      xe2x80x2      
xe2x80x83in which ODmax is the maximum optical density which may be measured,
Kxe2x80x2 is a constant,
ODmax and Kxe2x80x2 being determined by the following function f:       O    ⁢          xe2x80x83        ⁢    D    =            f      ⁡              (        V        )              =                            O          ⁢                      xe2x80x83                    ⁢                      D            max                                    (                                    K              xe2x80x2                        +            V                    )                    ·      V      
xe2x80x83in which V is the volume of the sample obtained by PCR subjected to step c) and OD is the optical density measured;
or by the following function g:       1          Q      ⁢              xe2x80x83            ⁢      D        =            g      ⁡              (                  1          V                )              =                            1                      α            xe2x80x2                          xc3x97                  1          V                    +              1                  O          ⁢                      xe2x80x83                    ⁢                      D            max                              
in which OD is the optical density measured, V is the volume of the sample obtained by PCR subjected to step c) and ODmax is the maximum optical density which may be measured;
the coefficient of amplification E1 of the DNA containing allele 1 being identical to the coefficient of amplification E2 of the DNA containing allele 2, for the different alleles of APOE.
The DNA amplified in step a) is, according to the invention, a cDNA comprising the allelic sequences of interest, obtained from mRNA by the usual technique of RT-PCR. The alleles are differentiated according to steps b) and c) described above, which demonstrate the restriction polymorphisms (RFLP).
The separation of the DNA fragments according to step c) may be carried out in particular by gel electrophoresis, preferably by polyacrylamide gel electrophoresis.
In the case of the APOE gene, the xcex53, xcex52 and xcex54 alleles may be characterized by restriction fragments of 91 bp, 83 bp and 72 bp, respectively.
The coefficient of proportionality A can therefore be calculated as being A=91/72 for the xcex53/xcex54 alleles, A being 83/72 for the xcex52/xcex54 individuals and A being 91/83 for the xcex52/xcex53 individuals. For the latter genotype, since the two xcex52 and xcex53 alleles give a restriction fragment length of 91 bp, the relationship AODallele 1+ODallele 2=OD91 bp is produced.
This method is in addition particularly simple. It is indeed sufficient either to present the value of the optical density (OD) as a function of the volume V of the sample, it being possible for the representative curve of the function f such that OD=f(V) to be plotted using the method of least squares, and to determine the ODmax and Kxe2x80x2 from this curve; or to present the value 1/OD as a function of the reciprocal of the volume of the sample, that is to say 1/V, it being possible for the representative curve of the function g such that       1          O      ⁢              xe2x80x83            ⁢      D        =      g    ⁡          (              1        V            )      
to be plotted by linear regression, the slope of the straight line obtained being equal to the reciprocal of xcex1xe2x80x2.
The subject of the invention is also a method for diagnosing Alzheimer""s disease comprising the determinations of the phase of the different polymorphisms xcex5, and Th1/E47cs and optionally of xe2x88x92491 AT and 1E1 which are capable of bringing about an increased risk of developing Alzheimer""s disease.
At the level of the population with the xcex53xcex54 genotype, the risk associated with the GT genotype is greater than that associated with the GG genotype. This observation can be explained by taking into account the phase of the polymorphisms Th1/E47cs and xcex52, xcex5 or xcex54 of APOE. Indeed, at the level of the heterozygotes GT and xcex53xcex54, the risk of developing the disease depends on the combination of the G allele and of the xcex54 allele on the same chromosome, thus determining the phase of the polymorphisms xcex5 and Th1/E47cs of APOE. Following this phase, two possibilities exist: (i) a higher level of expression of xcex54 (ii) a higher level of expression of the xcex53 allele. The first possibility would cause a physiological response in favour of the properties of the isomorph APOxcex54 and would explain a more marked effect at the level of the individuals carrying the xcex53xcex54 and GT genotypes. This difference in the level of expression linked to the phase will be true for all heterozygous genotypes. However, the risk associated with the G allele is probably attenuated because of the different combinations which are possible between the xcex5 and Th1/E47cs polymorphisms of APOE.
In accordance with the invention, the phase of the polymorphisms of APOE and of Th1/E47cs is studied in the following manner:
A fragment of 4600 bp containing the xcex5 polymorphisms of APOE and Th1/E47cs is amplified by PCR. This amplification is carried out using the kit: extend long template PCR system (Boehringer) with as sense oligonucleotide;
5xe2x80x2-GGGGGAGGTGCTGGAATCT-3xe2x80x2
and as antisense oligonucleotide:
5xe2x80x2-CAGATGCGTGAAACTTGGTGA-3xe2x80x2.
The product of amplification is then digested with the restriction enzyme Afl III, whose sole cleavage site on the amplified fragment makes it possible to differentiate the xcex54 allele from the xcex53 allele. After migration of the product of digestion on a 0.8% agarose gel, the DNA is transferred onto nitrocellulose membrane and fixed under UV. The discrimination between the T allele and the G allele is carried out by a protocol similar to that used for the ASO genotyping of this polymorphism.
Depending on the phases of the APOE and Th1/E47cs polymorphisms, it will be possible to define subgroups of sick subjects and to determine for each subgroup the optimum therapy. Likewise, the determination of the phase with other polymorphisms of the regulatory regions of the APOE gene capable of influencing (or otherwise) its expression, such as xe2x88x92491 AT described by Bullido et al., 1E1, APO CI, APO CII and D19S178 could also prove useful for the determination of subgroups of subjects at high risk of developing the disease.
Thus, it is known that subjects suffering from Alzheimer""s disease have a predisposition to respond to certain therapies, in particular to therapies of the cholinomimetic type depending on the type and the number of copies of the alleles of the APOE gene.
The subject of the invention is therefore also a method for identifying subjects suffering from Alzheimer""s disease who are capable of responding to a given therapy, for example of the cholinomimetic type, comprising:
a) the determination of the genotype of apolipoprotein E;
b) the determination of the genotype of the Th1/E47cs polymorphism; and
c) optionally, the determination of the phase of the polymorphisms of apolipoprotein E and of Th1/E47cs.
The different polymorphisms of APOE and of Th1/E47cs may be exploited to create animal or cellular models expressing APOE better or less well, used alone or in combination with the genetic factors capable of influencing the development of the pathology such as APP, PS1, PS2, and the like, or the other markers of Alzheimer""s disease such as abnormal phosphorylation of the protein Tau.
The subject of the invention is therefore also a transfection vector for eukaryotic cells comprising at least one allele of the APOE gene and one allele of the Th1/E47cs polymorphism and-optionally one or more other alleles of other genes or markers close to this gene, which are capable of modifying the risk of developing Alzheimer""s disease compared with a normal population.
These vectors may be used for the production of transfected eukaryotic cells or of transgenic animals.
Another subject of the invention therefore consists of the eukaryotic cells transfected by means of a vector as defined above or transgenic animals produced by means of such a vector.
The results of a study of the levels of expression of the mRNAs for the APOE gene in the brain will be given below.
Expression, in the Brain, of the APOE Gene in Patients Suffering from Alzheimer""s Disease Compared with Healthy Controls
1) Extraction of the RNA and amplification of the transcripts
The tissues were chosen in controls aged 65 years or over and in patients suffering from late onset Alzheimer""s disease, whose diagnosis has been confirmed by neuropathological examination, with heterozygous APOE genotypes.
The extraction of the RNA was carried out on samples of frontal lobes as described in J. Biol. Chem. 247, 4621-4627 (1972), or using an extraction kit (QIAGEN) and then the RNA was digested with DNase (Eurogentec). The RT-PCR was carried out with the aid of the primers described in J. Lipid. res. 31, 545-548 (1990). The reverse transcription reaction was carried out for 1 h 30 min at 37xc2x0 C., with the aid of primer F4 (5xe2x80x2-ACAGAATTCGCCCCGGCCTGGTA-3xe2x80x2) at 50 pmolar with 1 xcexcg of total RNA as template for the M-MLV reverse transcriptase, in accordance with the manufacturer""s instructions (Gibco/BRL). The PCR was carried out at 94xc2x0 C. for 10 minutes, followed by 30 cycles at 58xc2x0 C. for 1 minute, 72xc2x0 C. for 1 minute and 94xc2x0 C. for 1 minute.
The reaction volume of 25 xcexcl for the PCR contained the primer F6 (5xe2x80x2-TAA GCT TGC CAC GGC TGT CCA AGG A-3xe2x80x2) at 50 pmolar, the dNTPs at 0.5 mM, MgCl2 at 0.1 mM, triton X-100 at 0.1%, glycerol at 15% and Taq-Polymerase at 0.05 units (Eugentec).
The method of calculating the differential expression of the mRNAs is that described above.
The OD was estimated by the software (copyright)Imagemaster (Pharmacia).
The length of restriction fragments was determined at 91 bp for the allele xcex53, 83 bp for the allele xcex52 and 72 bp for the allele xcex54.
The value of the coefficient A corresponds for the genotypes xcex53xcex54 and xcex52xcex54 to A=91/72 and A=83/72 respectively. The value of the coefficient A corresponds for the genotypes xcex52xcex53 to A=91/83. The genotype xcex52xcex53 is characterized by the bands at 94 and 83 bp. In this case, given that the two alleles xcex52 and xcex53 give restriction fragments of 91 bp, AODxcex52=ODxcex53=OD 91 bp.
2) Results